Clinical Cytogenetics ch5 Flashcards
Cytogenetics
study of chromosomes within a cell
chromosomal abnormalities
-can be microscopic–> hard to detect
1 base change can be hard to see
-causes many syndromes
-collectively more than Mendelian single gene disorders
How to look for smaller scale changes in chromosomes? (2)
1) PCR: thermocycler–> sequencing= results
2) extract DNA send to a third party and get genome sequenced
Stats on cytogenetic disorders (3) –> Chromosomal abnormalities
!) 1% of live births
2) 2% of pregnancies when mom is >35 yrs
3) ~50% of 1st trimester spontaneous abortions
Cytogenetic Testing done in which situations (5)
1) mom over 35 yrs
2) growth/developmental delay
3) Still births/neomatal death
4) fertility problems
5) family history
6) neoplasia (cancer)
7) other (keep in mind that others can cause chromosomal abnormalities
cancer
uncontrolled cell growth causing a mass of cells
tumour can be benign or metastatic
benign tumour
not cancerous; can be removed and doesn’t spread to other parts of the body
malignant tumor
can invade tissues and organs
-also metastatic can break off into bloodstream
What materials are used for cytogenetic testing? LIST ONLY (for CGH/PCR) (5)
1) T-lymphocytes
2) White blood cells
3) Skin biopsy
4) bone marrow
5) fetal cells
T-lymphocytes
- short term
- limited number of divisions after extraction
- need lots of calls
Why do you use metaphase cells instead of interphase cells?
because chromosomes are condensed
easier to see
problem could be in cell division
White blood cells
- long term
- can divide more in lab= longer time to study.
- can be transformed into lymphoblastoid= cell lines that are potentially immortal
skin biopsy
samples of tissue
form fibroblasts that can be used for analysis
bone marrow
- hip bone because biggest bone and can get a big sample
- high proportion of dividing cells
Fetal cells
amniotic fluid/
chronic villi sampling–> can be studied directly
How do you distinguish between actual chromosomes?
by Size
If two chromosomes are almost the same size (ie 1 and 2) what do you look at to number them?
banding
banding patterns
characteristic dark and light stained regions
What stage of the cell cycle?
metaphase because more condensed easier to manipulate
-different stages= different banding patterns
heterochromatin
totally dark regions
genes are off in these regions
`
euchromatin
light bands
genes turned on here
G banding
- Giemsa banding
- G banding shows dark bands in AT rich areas (gene poor areas)
- promoters, centromeres; weaker regions 2 bonds btw AT vs 3 btw GC = HETEROCHROMATIN
R banding
R banding (reverse G banding) shows dark bands in GC rich areas (gene rich areas).
Q banding
- opposite of G banding
- bright Q bands = dark bands of G
C banding and which other type of banding….
and Q banding used to detect benign variants with differences in the amount or type of satellite DNA sequences at a certain location on the chromosome
which is the most common type of banding
G banding
p arm
short arm of a chromosome
q arm
long arm of a chromosome
numbering of a chromosome
increase from the centromere to the telomere
-centromere to the top
-and centromere to the bottom
(ends being higher in number
banding in prophase
longer; more darker bands than in metaphase and different sub-bands
banding in pro-metaphase
longer, and more lighter regions than in metaphase and different sub-bands
What can you detect from a standard karyotype?
6
- extra chromosomes
- deletion
- chromosome breaks (could lose large portions of chromosomes)
- translocations/ inversions= large pattern difference
- gender
- duplications
F.I.S.H
Fluorescence In Situ Hybridization
Technique used to hybridize DNA in FISH
1) get sample–> purify DNA
2) lyse cell& nucleus= chromosomes
3) put on a slide so they don’t move
4) denature them (chemically/heat)
5) put in an aqueous liquid to prevent desiccation and store
6) generate a probe –> need to know what we’re looking for and actually making
7) Hybridization (reannealing) probe to sample DNA
8) Microscope
Where to get a probe? (4)
- any piece of DNA in a tube that is cloned into a plasmid
- chop for a desired length using a restriction enzyme
- but it; make it by PCR
- isolate normal individuals DNA–> label that denature it and use it
Why is the signal more intense in the satellite probe?
because theres more DNA in a satellite DNA (telomeres/centromeres) than DNA at a locus
different probes with different colours
a) one green; 2 blue; one red
b) three blue; 2 green
c) three red; 1 blue and green
a) 46, XY
b) trisomy of chromosome 18 and female
c) trisomy of chromosome 21 and male
Small deletion detection
- FISH
- portion will be missing
- doesnt necessarily mean that theres a deletion; the chromosome could be scrambled ie) UV messing with thymine
DeGeorge Syndrome FISH
red probe to DNA deleted (22q11.2) supposed to be red dot in each one –> instead only one but control theres 2
FISH CHROMOSOME PAINTING
lots of probes from the same chromosome, each is labelled with the same florescent molecule therefore entire chromosome lighting up
Spectral karyotyping
-FISH used to paint each chromosome a different colour
-many probes for each chromosome
-Why? because easier for sorting, good for finding translocations (partial colours)
trisomies are clear
Clinical Diagnostic steps (9)
1) Patient has disorder–> dont know what it is access phenotypes first
2) Physician makes assessment
3) referral to speciallist
4) assessment of their own
5) family history
6) draw samples/ask physician to send you reports
7) prepare a karyotype (staining)
8) FISH (How to choose probe–> DR. some possibilities of disorders; limited number of probes needed)
9) nothing determined…..(what do we do now–> sequence entire genome –PCR based on phenotype inference
CGH (NAME?)
Comparative Genome Hybridization
CGH Definition
- probes that cover the entire ‘normal’ genome (or certain pieces) are fixed to a slide
- each ‘spot’ corresponds to a specific known position on a chromosome
Microarrays
- oligonucleotides (small DNA bits)
- whole genome on the chip with more than 1 copy
- label sample DNA–> complementary and read how many DNA bits have hybridized to the chip
Hybridize to slide
- small sample (from patient), fragment into small pieces and fluorescently label (red)
- take normal control DNA, fragment, fluorescently label (green)
- hybridize both to a slide
if quantity is same in patient and control?
normal
if quantity more in patient than control
extra chromosome/s
if quantity more in control than patient
patient is lacking some chromosomes
CGH Array from a computer gives a graph
ratio of 1.0=signal is equal to the control
trisomy for an autosome=1.5
monosomy=0.5
male=lower ratio of X; higher ratio for Y
female=higher ratio of X; lower ratio for Y
Chromosomal abnormalities
-changes in chromosome number (euploidy/aneploidy)
-changes in chromosome structure
(not entire chromosome)
what % of first-trimester abortuses are of a abnormal karyotype
50%
what fraction of mothers >35 are of a abnormal karyotye
1/50
what fraction of live births are of an abnormal karyotype
1/160
percentage of numerical abnormalities in first trimester abortuses
96%
percentage of mothers>35 with numerical abnormalities
85%
percentage of live births with numerical abnormalities
60%
Standardized Nomenclature for a deletion
46,X_,del (#p/q)
Standardized Nomenclature for a derivative
(der#)
der(1)
translocation chromosome derived from chromosome 1 and containing the centromere of chromosome 1
Standardized Nomenclature for a duplication (two types)
a) fragile site: 46,YorX,fra(X/Y)(p/q##.#)
b) isochromosome: 46,XorY,i(X/Y)(p/q#)
Standardized Nomenclature for an inversion
inv(#)(p/q#p/q#)
Standardized Nomenclature for a translocation
46,X_,t(#;#)(p/q#;p/q#)
46;XX,del(5p)
female with cri du chat syndrome, due to deletion of part of a short arm of one chromosome 5
46,Y,fra(X)(q27.3)
male with fragile X chromosome (fragile site)
46,X,i(X)(q10)
female with isochromosomes for the long arm of the X chromosomes
inv(3)(p25q21)
pericentric inversion of chromosome 3
46,XX,t(2;8)(q22;p21)
female with balanced translocation between chromosome 2 and chromosome 8, with breaks in 2q22 and 8p21
Monosomies are more deleterious than trisomies in live births
- complete monosomies are generally not viable except for monosomy X
- complete trisomies are viable for chromosomes 13,18,21,X and Y
Phenotype in partial aneusomies depends on
- the size of the unbalanced segment
- whether the imbalance is monosomic or trisomic
- which regions of the genome are affected and which genes are affected and which genes are involved
Mosaicism
person has a chromosome abnormality, the abnormality is in all of his/her cells
- when there are 2 or more different chromosomes that complement
- can be detected using FISH or CGH
inversions are either
pericentric or paracentric
an inversion: happens on a single chromosome and breaks in two places the segment btw is inverted (so ABCD into ACBD)
pericentric
- the risk of birth defects in offspring increases with the size of an inversion
- INCLUDES THE THE CENTROMERE
- BREAK IN EACH ARM
paracentric
very low risk of abnormal phenotype
- DOES NOT INCLUDE THE CENTROMERE)
- BOTH BREAKS HAPPEN IN THE SAME ARM
Euploidy
- exact sets of chromosomes
a) Triploid (3n): 69
b) Tetraploid (4n): 92 chromosomes –> spontaneous abortions
Aneuploidy
- common type of chromosomal disorder
a) Trisomy: one additional chromosome - most but only a few are viable (3,18,21, X&Y)
b) monosomy: one less chromosome -most lethal except X - 1 X= Turner’s Syndrome
Why only 13,18 and 21 trisomies?
- less genes than other chromosomes
- trisomic better gene imbalance than chromosome 19 which has many more genes
nondisjunction
- mitosis:the failure of sister chromatids to separate during and after mitosis
- meiosis: failure of homologous chromosomes to separate during and after meiosis
Causes of chromosome nondisjunction
A) the amount/location of recombination events during M1
-there are either too few or no recombinations
-recombination too close to centromere or telomere
B) premature separation of sister chromatids during M1, instead of M2
Abnormalities of chromosome Structure
A) less common than aneuploidies (1/375 live births)
B) large-scale rearrangements:
-spontaneous/induced: ionizing radiation/viral infection/chemicals in the environment
-all cells/only a subset of cells are infected:would have happened in the first division of the zygote
-stable/unstable: stable =can be passed on through mitosis; unstable=not
-balanced/unbalanced: loss of material; everything present in proper #; but just shifted around’
haploinsufficiency
inability of a single copy of the genetic material to carry out the functions of a single copy of the genetic material to carry out the functions normally performed by 2 copies
Terminal vs. Interstitial deletions
a) loss of the ends
b) interstitial: deletion in the middle of the arm
- terminal more likely than interstitial because terminal only needs one breakpoint; interstitial needs 2 breakpoints
deletion
- can occur in recombination
- can detect a deletion by CGH and FISH
duplication
- can be caused by unequal crossing-over
- generally less harmful than deletions
marker and ring chromosomes
-deletions of each end of chromosome and then reattachment
isochromsomes
-is a chromosome in which one arm is missing and the other is duplicated
Balanced Rearrangements
inversions and translocation all material is present just mixed up =no loss;no gain
Inversions
paracentric= no centromeres pericentric= includes the centromere
How to detect an inversion/translocation?
FISH/CGH/Chromosomal staining/PCR followed by sequencing
inversion: large than maybe chromosomal staining and then microscopy. PCR=maybe primers have to face each other= if they dont= fail
translocation: CGH: no loss or gain of material
FISH=YES because probe for the region testing for lightening up on a different chromosome= translocation
paracentric inversion heterozygote
no centromere; maybe depending on which gene was broken, resulting in a complicated looping. segregating and can break somewhere
-no impact on the individual but could affect the gametes
normal=ABCDE
deletion=ABCD
deletion=A
inversion product= ADCBE
common pericentric inversions
inv(3)(p25q21)
inv(8)(p23.1q22.1)
inv(9)(p11q12)
translocation
- exchange btw the arms of non-homologous chromosomes can be reciprocal/Robertsonian
- usually harmless for the carrier, can cause unbalanced gametes (due to breakage in a gene)
reciprocal translocation
type of rearrangement that results from the breakage of non homologous chromosomes with a reciprocal exchange
relocation of an oncogene
a gene that is not normally expressed is now becomes a housekeeping gene= which is expressed all the time in every gene
Robertsonian Translocation
-rare (1/1300)
-involves fusion of 2 acrocentric chromosomes
(acrocentric= centromeres at the end= p arm is gene poor
(chromosomes 13,14,15,21,&22)
insertion
- non-reciprocal
- segment from one chromosome is inserted into another; rare because it involves 2 breaks than a 3rd break to move into another chromosome
mosaicism
a) 2 or more populations of cells in one individual (usually aneuploid rather than structural)
- caused by bone marrow transplant etc..,
- causes: nondisjunction in an early meiotic division
- early= more cells that show an abnormality
Parent of Origin Effect
for some disorders, which disease phenotype is expressed depends on which parental chromosome is inherited how? genomic imprinting
Genomic imprinting
specific parts of specific chromosomes become imprinted (or marked) in the germline of one parent but not the other
- not a change in DNA only modification
- controls the expression of underlying genes after the formation of the zygote
- imprinting survives into adulthood
epigenetics
heritable changes in gene expression/function without changes to DNA sequences
a) DNA methylation
b) histone modification
DNA methylation
control for gene transcription
histone modification
acetylation (1 type)–> causes DNA to be compressed= no expression, modified acetylated= DNA available in euchromatin–> histone methylation= heterochromatin
Imprinting examples
a) Prader-willi Syndrome
b) Angelman Syndrome
Prader-willi Syndrome
obesity excessive eating small hands and feet short stature hypogonadism mental retardation
Angelman Syndrome
Unusual facial appearance
short stature
mental retardation
seizures
most common cause for both syndromes is the —–deletion
15q11-q13
if deletion inherited from FATHER
prader-willi
if deletion inherited from MOTHER
Angelman
